Over the past three decades, protein ubiquitination has emerged as an important post-translational modification with roles in a plethora of cellular processes including: proteolysis, gene expression, DNA repair, immune response, metabolism and cell-cycle regulation. Dysregulation of the ubiquitin proteasome system (UPS) has also been implicated in the pathogenesis of multiple human diseases including (but not limited to): cancer (Hoeller et al., Nat. Rev. Cancer (2006), 6, 776-788), viral infection (Gao et al., Can. J. Physiol. Pharmacol. (2006), 84, 5-14), metabolic or neurodegenerative disorders (Loosdregt et al., Immunity (2013), 39, 259-271; Rubinsztein et al., Nature (2006), 443, 780-786) as well as immune and inflammatory-related medical conditions (Wang et al., Cell Mol. Immunol. (2006), 3, 255-261; Corn J. et al., Nat. Struct. Mol. Biol. (2014), 21, 297-300; Nicholson et al., Cell Biochem. Biophys. (2011), 60, 61-68).
The approval and clinical success of the proteasome inhibitors Velcade® (bortezomib) and Kyprolis® (carfilzomib) for the treatment of mantle cell lymphoma and multiple myeloma (MM) have validated the UPS as a cancer target amenable for pharmacological intervention. Although effective, their clinical utility has however been severely limited due to poor selectivity and acute toxicity issues. By inhibiting the proteasome 26S subunit, the currently available proteasome inhibitors indiscriminately impair proteolysis in both cancer and normal cells and are therefore characterised by a low therapeutic index. To circumvent this issue, a promising alternative approach involves targeting the UPS upstream of the proteasome. Interfering with the ubiquitin (Ub) conjugation/deconjugation machinery, for instance, at the level of the ubiquitin specific protease (USP), should allow for the development of improved therapeutics with enhanced specificity and reduced toxicity profiles.
USPs are the largest sub-family of the deubiquitinating enzymes (DUBs) with over 60 family members reported to date (Komander et al., Nat. Rev. Mol. Cell Biol. (2009), 10, 550-563; Clague et al., Physiol. Rev. (2013), 93, 1289-1315). USPs are cysteine proteases that catalyse the removal of Ub from specific target substrates thus preventing their induced degradation by the proteasome, or regulating their activation and/or subcellular localization (Colland et al., Biochimie (2008), 90, 270-283; Nicholson et al., Cell Biochem. Biophys. (2011), 60, 61-68). It is now well established that USPs regulate the stability and activation of numerous proteins involved in the pathogenesis of human diseases including oncogenes and tumor suppressors. As such, USPs represent an emerging and attractive target class for pharmacological intervention.
Amongst all USPs, ubiquitin specific protease 7 (USP7—also known as herpes associated ubiquitin specific protease HAUSP) has attracted considerable attention due to implications in multiple oncogenic pathways (Nicholson et al., Cell Biochem. Biophys. (2011), 60, 61-68). USP7 plays a key role in regulating the ubiquitination and stability of the E3 ligase MDM2 (and human homolog MDM4) which in turn promotes the proteosomal degradation of the tumor suppressor p53 (Cummins, et al., Cell Cycle (2004), 3, 689-692; Cummins, et al. Nature (2004), 428, 486). Inhibition of USP7 reverses this process, restores p53 levels and ultimately results in anti-proliferative effects both in vitro and in vivo (Cheng et al., Cell Death and Disease (2013), 4, e867; Reverdy et al., Chem. Biol. (2012), 19, 467-477; Colland et al., Mol. Cancer Ther. (2009), 8, 2286-2295; Chauhan et al., Cancer Cell (2012), 22, 345-358). In addition to MDM2, USP7 has also been shown to mediate mono-deubiquitination of the tumor suppressor PTEN and transcription factor FOXO4, leading to their nuclear exclusion and respective inactivation (Song et al., Nature (2008), 455, 813-817; van der Horst et al., Nat. Cell Biol. (2006), 8, 1064-1073). Additional reported substrates of USP7 include (but are not restricted to): TSPYL5 (Epping et al. Nat. Cell Biol. (2011), 13, 102-108), the tumor suppressor p16 (Maertens et al., EMBO J. (2010), 29, 2553-2565), as well as various proteins involved in either genomic integrity or the DNA damage machinery such as: claspin, Tip60, UHRF1 or DNMT1 (Faustrup et al., J. Cell Biol. (2009), 184, 13-19; Dar et al., Mol Cell Biol. (2013), 33, 3309-3320; Qin et al., J. Cell Biochem. (2011), 112, 439-444; Du et al., Sci. Signal. (2010), 3, ra80; Nicholson et al., Cell Biochem. Biophys. (2011), 60, 61-68; Jackson et al., Nature Cell Biol. (2014), 16, 1016-1026). Finally, USP7 overexpression has been reported in multiple cancers including: prostate cancer, other haematological malignancies such as multiple myeloma, neuroblastomas and glioblastomas (Song, et al., Nature (2008), 455, 813-817; Cheng et al., Cell Death and Disease (2013), 4, e867; Chauhan et al., Cancer Cell (2012), 22, 345-358; Cheng et al., Oncology Reports (2013), 29, 1730-1736). Overexpression typically correlates with tumor aggressiveness and poor survival. USP7 inhibition may therefore have broad anticancer applications with potential use of small molecule inhibitors in mono- and/or combination treatment modalities. USP7 inhibitors may be particularly useful in disorders driven by overexpression of USP7, and/or in disorders driven by genetic (e.g. mutation, copy number variation) or epigenetic contexts.
In addition to cancer, USP7 has been shown to deubiquitinate and suppress the transcriptional activity of FOXO1 leading to suppression of gluconeogenesis in hepatocyte cell culture and animal studies (Hall et al., Mol. Endrocrinol. (2014), 28, 912-924). The involvement of USP7 in glucose metabolism may provide a strategy for clinical intervention in diabetes as well as in the treatment of other metabolic disorders including obesity. Furthermore, the connections and interactions of USP7 with viral proteins including for instance the EBNA1 protein of Epstein-Bar or the ICPO/VMW110 protein of herpes simplex type-1 viruses (Holowaty et al., J. Biol. Chem. (2003), 278, 29987-29994; Everett et al. J. Virol. (1999), 73, 417-426), strongly suggest that USP7 inhibitors may also be beneficial in the treatment of viral infections. Finally, the involvement of USP7 in regulatory T cell (Treg) function demonstrated in vitro and in vivo through modulation of the transcription factor FOXP3 stability (van Loosdregt et al., Immunity (2013), 39, 259-271), may open new therapeutic avenues for disorders characterised by inappropriate immune responses.
The established and growing connections between USP7 and numerous proteins involved in human disease indicate that small molecule inhibitors of USP7 may have broad therapeutic applications beneficial to human health. Small molecule USP7 inhibitors have been reported in the following patent applications: US 2008/0103149 A1, WO 2010/114881 A1, WO 2010/081783 A1, WO 2011/086178 A1, WO 2013/030218 A1, EP 2565186 A1, EP 1749822 A1, WO 2016/109515 A1, WO 2016/109480 A1, WO 2016/126929 A1, WO 2016/126926 A1, WO 2016/126935 A1, WO 2016/150800 A1, WO2017/158381, and WO2017/158388.